Batrachochytrium dendrobatidis (Bd) is the skin-invasive fungus that causes chytridiomycosis, a disease implicated as a proximate cause of global amphibian declines. While this pathogenic system is well-researched, our understanding of how environmental drivers regulate chytridiomycosis is incomplete. I aimed to develop and evaluate approaches to address how interactions between co-infecting Bd strains and host, pathogen and temperature affect Bd epidemiology. I also aimed to develop and evaluate methods to reduce the cost of qPCR-based detection in amphibians.

Two methods for reducing the cost of qPCR detection were identified: a reduced volume (10 μL) SYBR green qPCR on DNA extracted using either a CTAB extraction protocol or the standard extraction reagent, PrepMan Ultra. Reducing the volume of the SYBR green assay from 20 μL to 10 μL resulted in a slight reduction of assay sensitivity, from one zoospore in a sample to ten. However, the 10 μL assay performed well in validation against the standard Taqman qPCR assay, agreeing on positive or negative detection of Bd in 84.6% (CTAB) and 92.3% (PrepMan) of the samples tested. Therefore both of these protocols are suitable for use for detecting Bd in experimentally infected tadpoles; however the PrepMan extraction performed better than CTAB. The consumable costs of these protocols ranged from 38% (CTAB) to 42% (PrepMan) of the cost of the standard assay.

Hosts are commonly infected with multiple strains of a pathogen, and interactions between co-infecting strains can influence several aspects of disease epidemiology and evolution. Distinct strains of Bd have been identified but at present nothing is known about how they might interact. In Chapter 3 of this thesis I aimed to trial fluorescent probes for labelling Bd cells to distinguish between strains and track their fate. Two BODIPY and two CellTracker dyes (Molecular Probes, Invitrogen) were selected for trial. Both BODIPY dyes (558/568 and FL) and CellTracker orange CMTMR produced fluorescent Bd cells, but CellTracker green CMFDA did not. BODIPY 558/568 and FL were the most suitable for long-term tracking, at a concentration of 10 μM. At this concentration, BODIPY-labelled cells were brightly fluorescent for 12-16 days, distinguishable after being mixed together, and Bd growth was not inhibited.

In Chapter 4, I aimed to determine whether thermal patterns in Bd infection are explained by Bd thermal responses, or alternatively, by interacting host and pathogen thermal responses. Growth of Bd in culture was measured at the thermal optimum for Bd, 23°C, and at two sub-optimal temperatures, 15°C and 27°C. Litoria raniformis tadpoles were experimentally exposed to Bd at the three temperatures. The response of tadpole size, weight and developmental stage was measured and Bd infection was detected with qPCR. The growth response of Bd to temperature did not correlate with infection prevalence or abundance. Bd grew more rapidly at 23°C than at 15°C and 27°C. However Bd prevalence in tadpoles decreased linearly with temperature and was 0% at 27°C. There was no significant difference in mean Bd abundance at 15°C and 23°C. Neither did infection correlate with tadpole responses; tadpoles reached higher developmental stages and sizes at 23°C and 27°C than at 15°C. However, infected tadpoles were heavier and larger than uninfected conspecifics. Therefore it is likely that thermal patterns in Bd infection were determined by the interaction between tadpole and Bd responses to temperature.